ABSTRACT The discovery of NOTCH1 mutations in T-cell Acute Lymphoblastic Leukemia (T-ALL) patients raised hopes for pan-Notch inhibitors to treat this cancer. Unfortunately, in clinical trials, these drugs were too toxic because Notch has essential normal functions. Thus, the challenge is to selectively target Notch in T-ALL cells. Since Notch activity requires cofactors at its enhancers to create favorable cell type-specific ?chromatin contexts?, we envision that targeting these cofactors might avoid the intolerable effects of pan-Notch inhibition. Thus, our long-term goal is to understand the T-cell biology of Notch cofactors. For example, we discovered that the PIAS-like coactivator Zmiz1 is a direct cofactor of Notch1 that selectively promotes Notch activity at the T-cell specific Myc enhancer. Zmiz1 withdrawal or disrupting the Zmiz1-Notch1 interaction impaired Myc-dependent proliferation of pre-T cells and leukemic blasts. Importantly, ubiquitous inactivation of Zmiz1 did not have major effects on non-T cell tissues, suggesting more T-cell specific effects than Notch inhibition. For this proposal, we observed that the expression of Zmiz1 in pre-T cells dramatically increases from steady state levels during thymic regeneration. Accordingly, the Zmiz1-deficient pre-T cell defect is magnified 4-fold after irradiation compared to steady state. Thus, Zmiz1 is recruited to urgently restore T-cell immunity after cytoreduction. The Zmiz1 pathway might also have therapeutic utility as supraphysiological activation of Zmiz1 expanded pre-T cells and primitive early thymic progenitors (ETPs) in vitro. ETPs were expanded as Zmiz1 restrains Notch- induced T-cell differentiation signals, thereby protecting ETPs from excessive differentiation. Here, our objective is to understand these novel stage-specific roles of Zmiz1. Our hypothesis is that activating the Zmiz1 pathway induces stage-specific transcriptional programs that promote pre-T cell proliferation and ETP maintenance. To test this, we will determine how Zmiz1 is induced during thymic regeneration and how Zmiz1 facilitates transcription factor activities by remodeling chromatin. We will also determine how Zmiz1 manipulates cofactors and target genes to promote undifferentiated ETP proliferation. Finally, we will raise Zmiz1 signals to supraphysiological levels to enhance thymic recovery in vivo. Infection due to prolonged T-cell deficiency after various cancer therapies is a major clinical problem. Pan-Notch activation as a strategy to regenerate the T-lineage is problematic. Supraphysiological Notch activation depletes ETPs by promoting excessive T-cell commitment. In contrast, Zmiz1 preserves ETP cells, promotes proliferation, and by itself cannot induce leukemia. Thus, our project is significant because it will elucidate a direct Notch1 cofactor that drives leukemia and plays important stage-specific roles in enhancing early T-cell proliferation while restraining differentiation. We will learn new strategies to combat leukemia and promote thymic regeneration. Our project is innovative because it investigates the first instance that a Notch cofactor regulates thymic population dynamics in a manner that would promote balanced Notch-induced thymic regeneration.